Sialyltransferase st3gal6 as a marker for multiple myeloma

ABSTRACT

The present invention relates to the sialyltransferase ST3GAL6 for use as a biomarker for multiple myeloma, and especially as a marker for myelomas with inferior survival rates. The inventors have shown that glycosylation gene expression is dysregulated in Multiple Myeloma and that overexpression of the sialyltransferase ST3GAL6 is associated with inferior survival rates in patients.

FIELD OF THE INVENTION

The present invention relates to markers for Multiple Myeloma. Inparticular, the invention relates to the sialyltransferase ST3GAL6 foruse as a biomarker for multiple myeloma, and especially as a marker formyelomas with inferior survival rates. The inventors have shown thatglycosylation gene expression is dysregulated in Multiple Myeloma andthat overexpression of the sialyltransferase ST3GAL6 is associated withinferior survival rates in patients.

BACKGROUND TO THE INVENTION

Glycosylation is a stepwise procedure of covalent attachment ofoligosaccharide chains to proteins or lipids. Alterations inglycosylation are associated with malignant transformation, with growingevidence implicating the dysregulation of glycosylation genes(glyco-genes) in multiple myeloma (MM). P selectin glycoprotein ligand-1(PSGL1), recently reported to be important in MM cell adhesion andtrafficking is over expressed by MM cells. Importantly, the generationof functional selectin ligands requires post-translational modificationof scaffold proteins by glycosyltransferases and sialyltransferasesgenerating selectin ligands such as Sialyl Lewis X (sLex).

ST3Gal sialyltransferases are a family of enzymes that add sialic acidto the terminus of type I, II & III glycan chains. There are at least 20different human sialyltransferases, in four major sub-groups:-ST6GalNAcI to VI, ST3Gal I to VI, ST6Gal I to II and ST8Sia I to VI. Each ofthese sialyltransferase genes is differentially expressed in a tissue-,cell type-, and stage-specific manner to regulate the sialylationpattern of cells. They differ in their substrate specificity, tissuedistribution and various biochemical parameters: they specificallycatalyze the addition of sialic acid onto different sugars withdifferent sugar linkages (alpha 2, 3; alpha 2,6; alpha 2,8). In additionthe specificity of the sialyltransferase is determined by the structureof the underlying core sugar: e.g. ST3GAL6 adds sialic acid onto thegalactose part of an underlying core sugar structure, but only whengalactose is joined to N-acetyl glucosamine in beta 1,4 linkage whereasST3GAL1 will only catalyze the addition of sialic acid to galactosejoined to N-acetyl galactosamine in beta 1,3 linkage etc. ST3GAL6 canspecifically sialylate type II glycan chains (Galβ1-4GlcNAc) which formcritical components of sialyl Lewis X (sLe^(X)). sLe^(X) is thecarbohydrate ligand on glycoproteins and glycolipids which determinesselective binding activity for selectins. ST3GAL6 is a sialyltransferasewhich catalyses the transfer of sialic acid from cytidine 5-primemonophospho-N-acetylneuraminic acid (CMP-NeuAc) to terminal positions ofglycoprotein and glycolipid carbohydrate groups. Terminal Neu5Acresidues are key determinants of carbohydrate structures, such as theSialyl Lewis X. Sialyl Lewis X is a tetrasaccaride carbohydrate known toplay a vital role in cell to-cell recognition. Cellular trafficking inMM is mediated in the bone marrow by selectins such as P-selectin. Thecommon minimum binding determinant for all selectins is sialyl-Lewis X,the sialyation of this tetrasaccaride, mediated by ST3GAL6, is thereforepotentially critical to myeloma biology. It has recently been shown thatMM cells express high levels of P-selectin Glycoprotein Ligand-1(PSGL-1), leading to an increased interaction of MM cells withmicroenvironmental cells expressing P-selectin(1). Also inhibition ofP-selectin with the glycomimetic selectin inhibitor GM-1070(Glycomimetics) can sensitize MM cells to a successful treatment formyeloma called bortezomib, which highlights the potential role oftargeting glycosylation in MM.

Selectins are a family of cell adhesion molecules which have been shownto play an important role in cancer biology. There are three members ofthe selectin family: P-selectin expressed on activated platelets andendothelial cells, L-selectin present on leukocytes and E-selectinexpressed on activated endothelial cells. Besides the accepted roles ofselectins in physiological processes, such as inflammation, immuneresponse and hemostasis, there is accumulating evidence for thepotential of selectins to contribute to a number of pathophysiologicalprocesses, including cancer metastasis. Cancer cell interactions withselectins are possible due to a frequent presence of carbohydratedeterminants—selectin ligands on the cell surface of tumor cells fromvarious type of cancer. The degree of selectin ligand expression bycancer cells is well correlated with metastasis and a poor prognosis forcancer patients. There is emerging evidence that targeting selectinligands like P-selectin glycoprotein ligand1(PSGL1) in multiple myelomacan increase tumour cell sensitivity to current therapies. Untilrecently there were no reports of the effect of ST3GAL6 deficiency onleucocyte trafficking, however, it has very recently been reported thatP and E-selectin dependent leucocyte rolling was mildly reduced inST3GAL6-null mice and more severely in double deficient mice. There havebeen no reports of the effect of over expression or deficiency ofST3GAL6 in in vitro or in-vivo models of multiple myeloma.

Based on the above and on their work to date, the present inventorsbelieve that ST3GAL6 may have an important role in multiple myelomadisease biology.

It has been demonstrated in cell lines from multiple myeloma patientsthat ST3GAL6 is upregulated in comparison to non-malignant human celllines. It has also been demonstrated that ST3GAL6 is induced by hypoxiain myeloma cell lines MM1S, RPMI8226 and U266. Hypoxia has been shown tostimulate MM cell homing to the bone marrow microenvironment and hasbeen associated with multiple myeloma cell dissemination. Therefore itis possible that this gene may play a role in the mechanism of thesehypoxia-induced processes.

Using real time quantitative polymerase chain reaction in plasma cells(myeloma cells) selected from the bone marrow of patients with multiplemyeloma, it has been shown by the present inventors that the gene isupregulated in comparison to healthy controls. A trend was observedtowards higher expression of the gene in patients with more advancedmultiple myeloma and in patients with relapsed disease vs those who hada stable response to therapy. Therefore ST3GAL6 could serve as abiomarker in this disease with a potential prognostic significance.

ST3GAL6 codes for a transmembrane protein localized to the Golgiapparatus which has enzymatic activity catalyzing the transfer of sialicacid to terminal positions of glycoprotein and glycolipid carbohydrategroups. The present inventors have demonstrated expression of theprotein in bone marrow plasma cells from patients with multiple myelomaand demonstrated differential expression compared to normal healthy bonemarrow using immunohistochemistry and a specific antibody to ST3GAL6.

At the sugar level it has been demonstrated using biotinylated lectinbased flow cytometry that there is an increased level of alpha 2,3sialic acid on the surface of cultured myeloma cells. This has beenconfirmed using lectin based flow cytometry. Frithz et al (Eur. J. Clin.Oncol, vol 21, No. 8, pp 913-917, 1985) showed an increased serum levelof sialyltransferase in multiple myeloma patients. However noidentification of the actual sialyl transferase involved in the diseasewas undertaken in this paper. The present inventors are the first toimplicate a specific sialyltransferase in pathogenesis of multiplemyeloma.

The citation does not disclose the prognostic significance of anysialyltransferase or it's potential use as a biomarker of transitionfrom MGUS to MM with increasing levels associated with diseaseprogression.

FUCA1 is a gene that encodes the lysosomal enzyme alpha-L-fucosidase,which is involved in the degradation of fucose-containing glycoproteinsand glycolipids. Mutations in this gene are associated with fucosidosis(FUCA ID), which is an autosomal recessive lysosomal storage disease.

OBJECT OF THE INVENTION

It is an object of the present invention to provide a potentialbiomarker or drug target in multiple myeloma. It is also an object touse the biomarker to overcome current issues with therapy resistantdisease by enabling the correct treatment to be used for a given patientor by allowing the identification of new therapeutic agents.

It is a further object to allow the development of a disease specifictarget in multiple myeloma. In particular it is a further object to useST3GAL6 in the discovery of other biomarkers in multiple myeloma. It isanother objective to provide prognosticly significant markers, eitheralone or in combination, for Multiple Myeloma. A still further object isto provide a method of identifying therapeutic agents for the treatmentof multiple myeloma.

SUMMARY OF THE INVENTION

According to the present invention there is provided a method ofdiagnosing multiple myeloma in a patient comprising determining thelevels of ST3GAL6 in a sample obtained from the patient and comparingthe level determined with the level in a control sample, an increasedlevel relative to the control indicating the presence of multiplemyeloma in the patient. The control sample may be the level of ST3GAL6in at least one healthy individual. If a healthy individual is used as acontrol, then a diagnosis of MM is made on the basis of an increasedlevel of ST3GAL6 in the test sample. The skilled person will realisethat the method would also work using control levels from MM patients,in which case MM can be diagnosed if the levels of ST3GAL6 in the testsample are in the same range as those of MM patients.

The invention also relates to a method of determining the prognosis of amultiple myeloma patient comprising determining the levels of ST3GAL6 ina sample obtained from the patient and comparing the level determinedwith the level in a control sample, an increased level compared to ahealthy control is predictive of a reduced survival rate.

The sample tested may be a blood, plasma or serum sample or a tissuesample. The tissue sample could be a bone marrow sample.

The invention also provides methods, kits and uses of the markers FUCA1and ST3GAL1 in the diagnosis and assessment of prognosis of multiplemyeloma as described herein for ST3GAL6. The invention also providescombinations of one or more of ST3GAL6, FUCA1 and ST3GAL1 in suchmethods and uses.

The level of ST3GAL6, FUCA1 and ST3GAL1 may be determined byimmunohistochemistry, by RNA/DNA analysis, any form of immunoassay,including but not limited to an ELISA assay, a competitive or inhibitionELISA, a sandwich ELISA assay, a micro-array based assay, afunctionalised nanoparticle assay, other rapid assay platform such asQDots, F1 tags and electro sensors, or a flow cytometry assay or areal-time chip-based assays.

In another aspect the invention provides a kit for diagnosing multiplemyeloma in a patient comprising an antibody against ST3GAL6, or reagentsfor detecting the expression of DNA or RNA encoding ST3GAL6. Such a kitmay also be used for determining prognosis of a patient with multiplemyeloma comprising an antibody against ST3GAL6, or reagents fordetecting the expression of DNA or RNA encoding ST3GAL6.

Also provided is ST3GAL6 for use as a marker for diagnosing multiplemyeloma or for determining prognosis of a patient with multiple myeloma.

In yet another aspect the invention provides a method of screening atherapeutic agent for suitability for the treatment of multiple myelomacomprising testing a candidate therapeutic agent for the ability toreduce the expression of ST3GAL6 levels or activity of ST3GAL6 in amodel system, wherein a reduction of expression or activity of ST3GAL6indicates suitability for treatment of multiple myeloma.

Also provided is a method of reducing or alleviating multiple myelomacomprising administration of a modulator of ST3GAL6 in an amountsufficient to reduce ST3GAL6 expression or activity. The modulator maybe interfering RNA specific for ST3GAL6, a selectin inhibitor or aglycosyltransferase inhibitor. The invention also provides apharmaceutical composition comprising interfering RNA specific forST3GAL6, a selectin inhibitor or a glycosyltransferase inhibitortogether with a pharmaceutically acceptable carrier or excipient.

There appears to be deregulation of the ST3GAL6 gene as plasma cellstransition from the pre-myeloma phase to active myeloma with a clearincrease in ST3GAL6 mRNA in myeloma cells compared to their normalcounterparts. What leads to this deregulation is the subject of ongoinginvestigation but one factor the inventors have found to be associatedis hypoxia. Increased transcription leads to increased levels of theST3GAL6 sialytransferase enzyme, which can then be readily detected byimmunological techniques, including immunohistochemistry,immunocytochemistry, flow cytometry, etc. The serum of patients withhigh intracellular levels of ST3GAL6 may also contain increased levelsof the enzyme, which could be detected by ELISA. Ultimately, the highlevels of this enzyme catalyze the addition of excess alpha 2,3 sialicacid residues, leading to formation of high levels of sialyl Lewis Xantigen on the selectin ligands of affected cells (e.g. P-selectinligand or CD44). The increased expression of sialyl Lewis X as a resultof ST3GAL6 enzyme activity leads to increased interaction of myelomacells with selectins (both P and E) present on endothelial cells andbone marrow stromal cells, leading to increase cell trafficking,dissemination and drug resistance. The detection of high levels ofST3GAL6 is not diagnostic of myeloma but may help identify patients witha poorer prognosis and may also identify patients with earlier stagedisease (such as MGUS) who may actually be at higher risk of progressionto active disease than their counterparts with normal ST3GAL6. ST3GAL6overexpression may serve as a biomarker of response for treatment ofpatients with selectin inhibitors such that patients with ST3GAL6overexpression could eventually be targeted for treatment with selectininhibitors or other drugs interfering with the generation of selectinligands (such as glycosyltransferase inhibitors or RNA interferenceapproaches). ST3GAL6 overexpression has been associated with metastaticrisk in colon cancer and breast cancer but not in other haematopoieticmalignancies. It is quite likely it may play a role in otherhaematopoietic maligancies including lymphomas and acute leukaemia (bothmyeloid and lymphoid).

Gene Targeting

ST3GAL6 may serve as a target in multiple myeloma since reducedexpression of the gene may result in inhibition of selectin mediatedcellular adhesion and trafficking.

Targeting at the gene level could be mediated by a specific inhibitor ofST3GAL6 which may also have potential to induce down regulation of thegene.

Enzyme Targeting

Direct enzyme inhibition can be achieved by global inhibition ofsialyltransferases using fluorinated analogs of sialic acid as haspreviously been demonstrated in myeloid cells, where complete loss ofselectin binding and impaired leucocyte rolling was demonstrated. Thesialyltransferase inhibitor AL10 inhibits adhesion, migration, actinpolymerization and invasion of alpha-2,3-ST-overexpressing A549 andCL1.5 human lung cells by effectively attenuating total cell surfacesialylation. This compound is a lithocholic acid analogue derived fromsoyasaponin I by chemical synthesis in high purity and good yield. Asimilar approach to sialyltransferase inhibition could be adopted anddeveloped in multiple myeloma.

Sialic Acid Targeting

Significant reduction in rolling of sialidase treated neutrophils whenexposed to E and P-selectins has been demonstrated so direct globalinhibition of sialic acid on the surface of myeloma cells is likely havethe same effect with a resultant reduction in adhesion.

Measurement of Sialyltransferase in Blood and Bone Marrow

Measurement of serum sialyltransferase mean activities in patients withmultiple myeloma has previously been shown to be feasible. In untreatedpatients (as opposed to treated ones), a significantly higher serumsialyltransferase activity was shown among patients with stage IIImultiple myeloma in comparison to stages I and II, suggesting a linkbetween tumour burden and enzyme activity.

Detection Methods:

ELISA; Currently commercially available ELISA kits for the specificdetection of ST3GAL6 in patient serum could be employed to detect serumlevels as part of a prognostic or diagnostic test (MyBio Source catalog#MBS931514).

Flow Cytometry:

Flow cytometry analysis of CD138 microbead selected plasma cells fromblood and bone marrow of multiple myeloma patients can be used to detectthe presence of alpha 2,3 sialic acid on the cell surface. This can actas a surrogate marker of ST3GAL6 expression and a validation of the cellsurface glycosylation status. This widely available low cost techniquecan be performed immediately following bone marrow aspiration and wouldallow efficient establishment of the alpha 2,3 sialylation status ofindividual patients.

The inventors have performed this technique using multiple myeloma cellline RPMI8226 and demonstrated a significant shift in median fluorescentintensity for Maackia amurensis agglutinin (MAA) which is specific foralpha 2,3 bound sialic acids. RPMI cells were harvested incubated withMAA lectin for 30 mins at 4 degrees (200,000 cells/well in 100 uL).Following a washing step cells were incubated with streptavadin APC andincubated×30 mins at 4 degrees. MM cells were treated with mouse antihuman CD138 antibody or with an isotype control for 1 hour on ice.Expression of MAA lectin in CD138 positive cells was determined by flowcytometry analysis (BD Biosciences FACS Canto) and quantified as a ratioof the mean fluorescence intensity (MFI) of the detected target to theMFI of the isotype control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The ST3GAL6 gene is overexpressed in multiple myeloma celllines.

FIG. 2: ST3GAL6 gene expression is upregulated in response to hypoxia.

FIG. 3: Membrane proteins from multiple myeloma RPMI8226 cells areheavily sialylated consistent with increased activity of thesialyltransferase ST3GAL6.

FIG. 4: Membrane proteins from multiple myeloma MM1R cells are heavilysialylated consistent with increased activity of the sialyltransferaseST3GAL6.

FIGS. 5 a and b: ST3GAL6 protein can be detected by immunohistochemistryin bone marrow plasma cells from patients with multiple myeloma. FIG. 5a is, from a patient with multiple myeloma, while FIG. 5 b is from anormal bone marrow

FIG. 6: Increased gene expression of ST3GAL6 is associated with inferioroverall survival in the UK MRCIX Myeloma clinical trial.

FIG. 7: ST3GAL6 protein is strongly expressed by multiple myeloma celllines.

FIG. 8 a: QPCR data for MM1s cells infected with a silencing lentivirusconstruct to ST3GAL6 in comparison to the ST3GAL6 mRNA level in ascrambled control, (b) the reduction of the mRNA level in the RPMI-8226cell line. A1, A2, A3 and A4 represent different lentivaral constructs.

FIG. 9 Western blots of shST3GAL6 multiple myeloma cell lines.

FIG. 10. Adhesion of shST3GAL6 myeloma cell lines to fibronectin.

FIG. 11. Adhesion of shST3GAL6 myeloma cell lines to primary bone marrowstromal cells taken from the bone marrow of myeloma patients.

FIG. 12. Transwell assay assessing the ability of shST3GAL6 cells tomigrate to the chemokine SDF-1α migration.

FIG. 13. Average bioluminescent intensity signal in SCID-Bg mice(N=7/group).

FIG. 14. In-vivo confocal imaging performed in SDID-Bg xenograft miceskull bones. Red represents vessels stained with Evans Blue dye, greenrepresents myeloma cells (MM1s-GFP+ cell line, (n=3/group,representative images shown from two mice).

FIG. 15: Myeloma cells express sialytransferases and sLex.

FIG. 16. Combining gene expression data of FUCA1 with expression ofeither ST3GAL6 or ST3GAL1 or both, patients with a particularly pooroutcome are shown in red and black lines.

DETAILED DESCRIPTION OF THE DRAWINGS

Materials and Methods

Real time quantitative PCR (RT_PCR) in the MM cell lines RPMI8226 andMMIR and in primary MM patient CD138 positive cells was used to examineexpression of the ST3GAL6 gene. The prognostic significance of theST3GAL6 gene was analyzed using Kaplan Meier survival estimates.Membrane protein extracts from MM cell lines were applied directly tolectin microarrays following fluorescent labelling to generate cellsurface glycan profiles. Protein expression was assessed byimmunohistochemistry (IHC) on primary MM bone marrow sections from MMpatients and healthy controls. Lectin based flow cytometry was carriedout to assess for the binding of sialic acid lectins to MM cell lineRPMI8226.

CD138 Cell Isolation

Bone marrow aspirates were obtained from multiple myeloma patients atGalway University Hospital following informed consent. The mononuclearfraction of the bone marrow aspirate was isolated using Ficoll Paque™Plus (STEMCELL Technologies) and high speed zero brake centrifugation.PC isolation from mononuclear cell fraction was performed byimmunomagnetic bead selection with monoclonal mouse antihuman CD138antibodies using the AutoMACs automated separation system(Miltenyi-Biotec, Auburn, Calif.). PC purity of more than 95%homogeneity was confirmed by 2-color flow cytometry using CD1381/CD452and CD381/CD452 criteria (Becton Dickinson, San Jose, Calif.).

Real Time Quantitative Polymerase Chain Reaction (RT_PCR)

The mRNA expression of ST3GAL6 was detected by RT-PCR in indicated MMcell lines and in sorted CD138+ cells from MM patients with the forwardprimer TTG CCT CTC TGC TGA GGT TT and the reverse primer CCT CCA TTA CCAACC ACC AC The expression of GAPDH or 18S was detected with theirrespective primers. RT-PCR amplification was carried out with 100 ng ofcDNA in a 10-μl reaction mixture containing 5 ul SYBR green mix(Bio-Rad, Mississauga, ON) and 0.2 ul qPCR primers (SA Bioscences,Frederick, Md.). Data quantification was carried out by the deltadeltaCTmethod.

Lectin Array

Glycan-binding proteins (Lectins) conjugated to microarrays were used toprobe compositions and differential presence of glycan residues onglycoconjugates and cells. The glycan samples were fluorescently taggedprior to hybridization step to co-incubate glycan samples and lectins.After hybridization, slides were washed extensively and scanned in amicroarray scanner. The lectin-glycan binding data is analyzed usingcommercially available software.

Sialyltransferase Serum Assay

This assay employs the quantitative sandwich enzyme immunoassaytechnique. Antibody specific for ST3GAL6 has been pre-coated onto amicroplate. Standards and samples are pipetted into the wells and anyST3GAL6 present is bound by the immobilized antibody. After removing anyunbound substances, a biotin-conjugated antibody specific for ST3GAL6 isadded to the wells. After washing, avidin conjugated HorseradishPeroxidase (HRP) is added to the wells. Following a wash to remove anyunbound avidin-enzyme reagent, a substrate solution is added to thewells and color develops in proportion to the amount of ST3GAL6 bound inthe initial step. The color development is stopped and the intensity ofthe color is measured.

Immunohistochemistry

To detect ST3GAL6, BM aspirates from 55 MM patients and 3 healthysubjects were rinsed with PBS, fixed with 4% formaldehyde in PBS,dehydrated with ethanol, embedded in paraffin, and sectioned. Tissueswere then immunostained with mouse anti-human ST3GAL.

Knockdown of ST3GAL6 in Myeloma Cell Lines;

Green Fluourescent Protein (GFP) Luc⁺-MM1s cells have been generatedusing lentiviral infection and cultured at 37° C. in RPMI 1640 mediumcontaining 10% FBS (Sigma-Aldrich), 2 mM L-glutamine, 100 U/mL ofpenicillin, 100 μg/mL of streptomycin (Invitrogen) and 100 μg/mLGeneticin. Plasmids were routinely amplified in the Escherichia coliDH5_strain and isolated from cultures by using the Qiagen Plasmid SpinMidiprep Kit (Qiagen, Valencia, Calif.).

Scrambled shRNA control plasmids can be generated using the same methodsused to generate lentivirus. Scrambled lentivirus is used to infect MM1scell to act as a control in these experiments. Stable knockdown ofST3GAL6 can be achieved in these cells using a plasmid generatedlentiviral vector which infects the MM1s cells.

Interfering RNA Screening

IRNA screening is performed in cultured MM cell lines as a lethalitystudy. Using technology available from the Broad Institute Boston Mass.,USA siRNAs targeting individual human silayltransferase genes arepreprinted on 384-well plates alongside staggered negative and positivecontrol siRNA. Primary screening experiments are conducted in duplicateon separate occasions for several cultured MM cell lines. For eachexperiment, plates preloaded with siRNA and frozen were thawed at roomtemperature and 20_L/well-diluted Lipofectamine 2000 or Dharmafect 3solution is added to the relevant wells. After 30 minutes, MM cells areadded to each well. Cell viability is determined at 96 hours byCellTiter-Glo luminescence assay read on a BMG Polarstar machine usingexcitation 544-nm/emission 590-nm filters. Surviving knock down cellscan be analysed by the following methods.

Cellular Adhesion Assays

BMSCs are cultured overnight to confluence in 96-well plates (5_(—)103cells/well) before initiating the adhesion assay. MM1s cells areserum-starved for 3 hours, prelabeled with GFP, added to the BMSCs(1_(—)105 cells/well), and allowed to adhere for 2 hours at 37° C.Nonadherent cells can be aspirated away, the BMSCs washed, andfluorescence intensity measured using a fluorescent-plate reader(Ex/Em_(—)485/520 nm).

Transendothelial Migration and Chemotaxis of MM Cells

HUVECs (5_(—)103 cells/basket) are incubated overnight in the upperchamber of 8-micron pore filters (Costar; Corning) before performing theadhesion assay. MM1s cells are serum-starved for 3 hours and then addedto the upper chamber of the basket (2_(—)105 cells/well), and left tomigrate for 4 hours at 37° C. toward the lower chamber, which contains 0or 30 nM of SDF1_. This analysis is performed separately for ST3GAL6knockdown cells vs scrambled control.

Assessment of Multiple Myeloma Tumour Burden in SCID Mice;

This is carried out according to methods developed by David Scadden'sgroup (13). In vivo confocal microscopy is used to test the homing ofMM1s cells to the BM in vivo and to assess the tumour burden ofGFP-labelled MM1s cells (1_(—)106) injected into anesthetized SCID mice.In some experiments mice may be pretreated with vehicle or a drug ofinterest 1 hour before the cell injection. A skin flap is made in thescalps of the mice to expose the underlying skull surface and Evan'sblue dye is injected intravenously immediately before imaging tovisualize blood vessels. High-resolution images with cellular detail arecaptured 30 minutes after cell injection through the intact mouse skullat depths of up to 250_m from the surface of the skull using a30_(—)0.9-numerical aperture water-immersion objective lens (Lomo).Multiple imaging depths were acquired and a maximum intensityz-projection is performed in ImageJ software to merge the images. GFPlabelled MM cells and Evan's blue dye are excited with 491-nm and 638-nmsingle-photon lasers and detected via 528/19-nm and 680/25-nm bandpassfilters, respectively.

Materials and Methods ST3GAL6 Study:

In-Vitro Studies:

Cell lines & Reagents: The RPMI-8226 cell lines were purchased from ATCC(Manassas Va.) cultured in RPMI 1640 (Sigma Chemical, St. Louis, Mo.)containing 10% foetal bovine serum, 2 mmol/L L-glutamine (LifeTechnologies, Grand Island, N.Y.), 100 units/mL penicillin, and 100Ag/mL streptomycin (Life Technologies).GFP-Luciferin-Neo MM1s cells weregenerated using a lentiviral infection method as described [1]. PrimaryMM cells were obtained from bone marrow samples from patients usingCD138+ microbead selection (Miltenyi Biotech, Auburn Calif.) aspreviously described [2]. Bone marrow stromal cells (BMSC's) wereobtained from healthy donor and MM patient fresh bone marrow samples andisolated as described [3]. BMSC's were then cultured in DMEM (SigmaChemical, St Louis, Mo.) supplemented with 10% heat-inactivated foetalbovine serum, 100 units/Ml penicillin, 10 Ag/mL streptomycin (LifeTechnologies; 0.01%), and 2 mmol/L L-glutamine (1%; Life Technologies).Informed consent was obtained from all patients in accordance with theDeclaration of Helsinki Approval of these studies was obtained by theDana-Farber Cancer Institute Institutional Review Board. Bortezomib wasobtained from Millennium Pharmaceutical (Cambridge, Mass.), and SDF-1was obtained from R&D Systems (Minneapolis, Minn.). shRNA meditatedST3GAL6 gene silencing: To determine the role of ST3GAL6 in MM biologywe established ST3GAL6 knockout RPMI-8226 and MM1s-GFP-Luc cell linesusing a lentiviral system as previously described [4]. A plasmid basedsystem was used to achieve knockdown of ST3GAL6 in the MM cell lines.Lentiviral ST3GAL6 shRNA and non target scrambled control shRNA wasproduced in HEK293T packaging cells, concentrated at different MOIs andthen individually added into MM-cell suspensions in the presence of 8mg/mL polybrene and transduced for 24 hours followed by selection inpuromycin (2 mg/mL; Invitrogen). The efficiency of ST3GAL6 knockdown wasassessed by flow cytometry and RT-PCR using the following primersForward Primer; TTG CCT CTC TGC TGA GGT TT Reverse Primer; CCT CCA TTACCA ACC ACC AC [5]. The resultant stable ST3GAL6 knockdown (shST3GAL6)cell line is compared to the scrambled control cell line in allsubsequent functional assays. The sense and antisense oligonucleotidesequence for construction of ST3GAL6 shRNA were as follows: clone no10402; [NM_(—)006100.2-1332s1c1, target sequence CCTTTGCACTACTATGGGAAT(A2), NM_(—)006100.2-1110s1c1 target sequence CCAGCCTTAAACCTGATTTAT(A3)]

Adhesion assays: Assays for adhesion of MM cells to normal and MM BMSC'swere performed using 96-well plates (5×10³/well). Normal or MM BMSC's(5,000/well) were seeded in 96-well plates overnight in supplementedDMEM media to establish a confluent monolayer. MM cells were serumstarved overnight, prelabelled with calcein AM and added to the BMSC'sand allowed to adhere for 2 hours at 37° C. Nonadherent cells wereaspirated off, BMSCs were washed with PBS, and fluorescence intensitywas measured using a fluorescent-plate reader (Ex/Em_(—)485/520 nm).Fibronectin adhesion assays were performed using an in vitro adhesionassay coated with fibronectin following the manufacturer recommendations(EMD Biosciences, San Diego, Calif.) as previously described [6]. Bovineserum albumin (BSA)-coated wells served as negative controls. In some ofthe above experiments MM cells were pre-incubated with increasingconcentrations of bortezomib (0, 2.5, and 5 nM) prior to assessment ofadhesion.

Migration assays: Migration assays were performed as previouslydescribed [6] [7]. Migration ability of MM scrambled control cells vs.shST3GAL6 cells was assessed using a transwell migration assay plate(pore size 0.8_m; Corning Life Sciences, Acton, Mass.) according to themanufacturer's instructions. MM cells were serum starved for 4 hours andfollowing calcein AM labelling were then placed in the upper migrationchambers. SDF-1_(30 nM) was added to 500_uL of RPMI 1640 in the lowerchambers. After 4 hours at 37° C. cells that migrated to the lowerchambers were counted on a fluorescent plate reader (Molecular Devices).

Immunoblotting: Immunoblotting was performed as previously described[8]. Briefly, whole-cell lysates were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred topolyvinyldene fluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules,Calif.). The antibodies used for immunoblotting included anti-ST3GAL6produced in rabbit (Sigma-Aldrich, Mo., USA) and anti-Actin (CellSignaling Technology)

Immunohistochemistry: To detect ST3GAL6, BM aspirates from 50 MMpatients and 10 healthy subjects were rinsed with PBS, fixed with 4%formaldehyde in PBS, dehydrated with ethanol, embedded in paraffin, andsectioned. Tissues were then immunostained with mouse anti-human ST3GAL6antibody and mouse-antihuman-CD138.

In-Vivo Xenograft Models

Animals:Approval of these studies was obtained by the Dana-Farber CancerInstitute and Massachusetts General Hospital Institutional Animal Careand Use Committees. Female, 6 to 7 weeks old, severe combinedimmunodeficient beige (SCID-Bg) mice were obtained from TaconicLaboratories. Anesthesia was performed by intraperitoneal injections ofketamine (Bedford Laboratories, Bedford, Ohio)/xylazine (LloydLaboratories, Shenandoah, Iowa) at 80 mg/kg/12 mg/kg body weight priorto in-vivo imaging sessions. Following this mice were killed byinhalation of CO2.

Xenograft models: Tumors were established in mice using MM1s-GFP-Luccells (5_(—)10⁶/mouse) which were injected into the tail vein of 12SCID-Bg mice (n=6/group).

BLI: To detect tumor burden on a weekly basis following injection micewere injected with 75 mg/kg luciferin (Xenogen, Hopkinton, Mass.) andimaged for bioluminescence 5 minutes after the injection. The home-builtbioluminescence system used an electron multiplying CCD (AndorTechnology, Belfast, United Kingdom) with an exposure time of 15seconds, an electron multiplication gain of 500-voltage gain_(—)200,5-by-5 binning, and background subtraction. Images were analyzed withthe use of ImageJ software (National Institutes of Health, Bethesda,Md.).

In-vivo confocal: Using the above SCID-Bg xenograft mice homing of MMcells to distant bone marrow niches was tracked in vivo, by using invivo confocal microscopy [9]. Briefly, MM cells homing to the BM wereimaged in vivo using a Zeiss 710 confocal system (Carl ZeissMicroimaging, Jena, Germany) on an upright examiner stand with a customstage. A skin flap was made in the scalp of the mice to expose theunderlying dorsal skull surface. High-resolution images with cellulardetail were be obtained through the intact mouse skull at depths of upto 250 μm from the surface of the skull using a 10×0.45 NA Plan-Apoobjective (Carl Zeiss Microimaging). Multiple imaging depths wereacquired, and a maximum intensity z-projection was performed in Image Jto merge the images. GFP was excited with the 488 nm line on an Argonlaser. Blood vessels were imaged using Evans Blue (Sigma-Aldrich, St.Louis, Mo.) excited with a 633 nm laser. Emission signals were becollected by the Zeiss internal confocal Quasar detectors.

Results

The sialyltransferase ST3GAL6 (ST3 beta-galactosidase,alpha-2,3-sialyltransferase 6), which plays a critical role ingeneration of functional selectin ligands such as sLex, was upregulatedin MM (fold change=2.67) and smoldering MM (fold change=2.22) but wasabsent in MGUS (Monoclonal gammopathy of undetermined significance).Patients from GSE24080 (n=509) were stratified into two groups based ontheir expression intensities for ST3GAL6. Patients with highernormalized intensity values corresponding to probeset ID 210942_s_at(ST3GAL6) were found to have a lower median survival compared to theirlower expressing counterparts. The difference in survival observed was5.13 months (p<0.001 CI 1.3, 8.9). The association with reduced survivalwas independently verified in the MRC Myeloma IX microarray dataset(n=260). In this dataset using the median expression of ST3GAL6 as acutoff there was a statistically significant reduction on overallsurvival with higher expression of ST3GAL6 (median OS 35.7 vs 48 months,log rank test p=0.04).

RT-PCR analysis validated the over expression of ST3GAL6 in MM celllines and primary samples compared to healthy controls. We observed atrend for higher fold changes for ST3GAL6 in samples from patients withrelapsed/refractory disease compared to those with responsive disease.IHC for ST3GAL6 on primary bone marrow sections from MM patients (n=27),demonstrated specific Golgi staining compared to controls. Consistentwith the over expression of ST3GAL6 lectin microarray analysis ofmembrane protein extracts from MM cell lines RPMI8226 and MMIR showedbinding to sialic acid-specific lectins Maackia amurensis agglutinin(MAA), which is specific for α2-3 bound sialic acids, and Sambucus nigra(SNA) which is specific for α2-6 bound sialic acids. This pattern wasconfirmed using biotinylated lectin based flow cytometry, whichdemonstrated a shift in allophycocyanin (APC) median fluorescenceintensity for these lectins on RPMI8226 cells.

As shown in FIG. 1 quantitative PCR was used to detect mRNA for ST3GAL6in CD138 plasma cells from normal donors as well as 5 different multiplemyeloma cell lines. FIG. 2 demonstrates the effect of hypoxia (cellsgrown in hypoxic chamber with 1% O₂) on the gene expression of ST3GAL6as assessed by quantitative PCR on 3 different multiple myeloma celllines. FIGS. 3 and 4 demonstrate the pattern of lectin binding tomembrane proteins from the multiple myeloma cell lines RPMI8226 and MM1Rusing a lectin array. This pattern of lectin binding indicates that themembrane proteins are heavily sialylated.

FIGS. 5 a and b show co-expression of CD138 and ST3GAL6 byimmuno-histochemistry. FIG. 5 a, from a patient with multiple myelomashows an excess of bone marrow plasma cells with expression of ST3GAL6in CD138 positive cells. On the other hand, FIG. 5 b, from a normal bonemarrow, shows absence of ST3GAL6 in CD138 cells, which are present atlow frequency in the marrow. Increased gene expression of ST3GAL6 isassociated with inferior overall survival in the UK MRCIX Myelomaclinical trial, as shown in FIG. 6 which shows two separate Kaplan Meiersurvival curves for the 259 patients treated on the UK MRCIX Myelomaclinical trial according to whether they had high or low levels ofexpression of ST3GAL6. The 129 H patients had expression values abovethe median while the 130 L patients had expression values below themedian. The difference in survival between the 2 groups was comparedusing the logrank test and is statistically significant. In patientswith the highest levels of gene expression of ST3GAL1 (top 25%) there issignificantly worse progression free survival. The highest levels ofST3GAL1 gene expression are seen in patients with recognised poor riskchromosome abnormalities, including t(4; 14) translocation andhypodiploidy, identification of which are a standard part of the work upof myeloma patients.

FIG. 7 shows protein expression of ST3GAL6 by Western blotting in normalCD138 bone marrow derived plasma cells compared with ST3GAL6 proteinexpression in 3 multiple myeloma cell lines. While no appreciable bandcan be seen in the normal plasma cells, a strong band is seen in all 3cell lines.

To directly identify the biological role of ST3GAL6 in myeloma,lentivaral stocks were produced from hairpin-pLKO.1 plasmids andtransfected into myeloma cell lines MM1s-green fluorescentprotein-luciferase positive (GFP-Luc) and RPMI-8226. The MM1s-GFP-Luccell line was chosen in order to create a stable shST3GAL6 cell linethat was amenable to later in-vivo imaging and Confocal imaging. TheRPMI-8226 cell line was chosen as a representative cell line of multiplemyeloma which had not been previously manipulated. As FIG. 8 a shows,QPCR data for the MM1s cell line demonstrates the reduced level of mRNAdetectable in cells that had been infected with a silencing lentiviralconstruct to ST3GAL6 in comparison to the ST3GAL6 mRNA level in ascrambled control. FIG. 8( b) demonstrates the effective reduction ofthe mRNA level in the RPMI-8226 cell line.

Western blots demonstrating the reduced level of protein detected in theshST3GAL6 stable cell lines in comparison to respective scrambledcontrols are shown in FIG. 9. In-vitro functional studies wereundertaken to determine the role of ST3GAL6 on MM-cell adhesion,migration and survival. The results of the adhesion analysis tofibronectin are shown in FIG. 10. MM cells were serum starved overnight(5×10⁵/ml) and then incubated with calcein-acetoxymethyl ester (AM) for30 mins at 37° C., washed and re-suspended in serum free clear RPMImedia. 100 ul of cells were added to fibronectin coated 96-well plates(Calbiochem ECM Cell Adhesion Assay) for 1 hour at 37° C. After a gentlewas in PBS the fluorescence was read using a fluorescent plate reader(ex/em 485/520 nm).

The results of the adhesion analysis to primary bone marrow stromalcells are shown in FIG. 11. MM cells were serum starved overnight and(5×10^(5/ml)) following this were incubated with calcein-AM as describedfor 30 mins at 37° C., washed and re-suspended in serum free clear RPMImedia. 100 ul of cells were added to BMSC coated 96-well plates(3×10⁴/well) for 2 hours at 37° C. After a gentle wash with PBS thefluorescence was read using a fluorescent plate reader (ex/em485/520nm).Results of the migration studies are shown in FIG. 12. Formigration studies cells (5×10⁵) were serum starved×4 hours in clearserum free RPMI media, labelled with calcein-AM as described and addedto the upper wells of transwell plates (Costar Corning pore size 0.8 μm)with serial concentrations of SDF-1α (0-30 nM) added to the 500 ul ofRPMI 1640 serum free media in the lower chambers. After 4 hours at 37°C. the upper chambers were removed and cells that had migrated to thelower chambers were counted on a fluorescent plate reader.

In-vivo studies in the SCID-bg xenograft mouse model of MM wereconducted to assess the effect of shST3GAL6 on myeloma xenograft tumourgrowth in-vivo compared with scrambled control cells were conducted.FIG. 13 shows bioluminescent imaging of SCID-Bg mice weeks 1-4 post1×10⁶ MM1s-GFP-Luc positive cells/mouse injected IV via tail vein.Scrambled control group received cells that were infected with astandard scrambled control lentivirus (shGFP). Animals were imaged eachweek for monitoring of tumour development 5 minutes postintra-peritoneal injection of luciferase enzyme as per standardprotocol. At week four animals were sacrificed for confocal imaging.In-vivo confocal imaging was performed on SDID-Bg xenograft mice skullbones. In FIG. 14 red represents vessels stained with Evans Blue dye,green represents myeloma cells (MM1s-GFP+ cell line) and can be taken torepresent tumour burden in the skull bone marrow. Images taken 4 weeksfollowing injection of 5×10⁶ cells injected intravenously via tail vein.Mouse A was injected with scrambled control cells, mouse B was injectedwith MM1s-GFP+ cells that had been infected with shRNA for ST3GAL6resulting in a stable knockdown of this gene in the cell line. A numberof other glycosylation genes are differentially regulated between normaland malignant plasma cells in MM. The prognostic significance of certaingenes was analysed using Kaplan Meier survival estimates for progressionfree survival and overall survival. Low expression (lower quartile byGEP) of the gene FUCA1, was linked to inferior outcome (median OS 44.v.38 mos, p=0.025). On multivariate analysis low FUCA1 expression wasindependent of other important prognostic factors. FUCA1 expression wasnot copy number sensitive and there was no correlation with methylationstatus in the MRCIX dataset.

MM cell lines show low levels of FUCA1 transcripts by QPCR. The OPM2cell line, which contains t(4:14) showed increased ST3GAL1 transcriptsrelative to NBM. High expression (top quartile) of the sialytranserasegene ST3GAL1 also showed a trend towards inferior OS (median survival 35mos.v. 45 mos, p=0.07) with significantly reduced PFS (19.v. 14 mos,p=0.015),Increased expression of ST3GAL1 correlated with the presence oft(4;14) (p=0.009), del13q (p=0.001), +1 q (p=0.01) and hypodiploidy(p=0.00001).

As shown in FIG. 15 myeloma cells express sialytransferases and sLex/a.Since FUCA1 participates in N-glycan degradation with removal of fucoseresidues, this raises the possibility that a reduction in FUCA1 may leadto excessive fucosylation of cancer related glycans involved in adhesionand trafficking, such as sLex. Lectin arrays of MM cell lines revealedin high levels of a-1,3/a-1,6 linked fucose, as determined by binding ofAleuria Aurantia Lectin (AAL) (data not shown). The HECA-452 antibodyrecognizes a functional trisaccharide domain shared by sialyl Lewis aand sLex and known to bind to E-selectin. Preliminaryimmunohistochemistry revealed increased HECA452 staining (D) in bonemarrow samples, which had both strong ST3GAL6 (A)+/ST3GAL1 (B) and lowFUCA1 staining (C).

Combining gene expression data showed that reduced expression of FUCA1with increased expression of either ST3GAL6 or ST3GAL1 or both,identified a subgroup of patents (18%) in MRC Myeloma IX withparticularly poor outcome (red and black lines), as shown in FIG. 16.Similar results where found in independent datasets with 17% of patientsaffected.

Conclusions

The sialyltransferase ST3GAL6 is differentially regulated in all stagesof MM with potential effects on MM biology and survival. Upregulation ofST3GAL6 may play an important role in MM cell trafficking and in thisanalysis is associated with inferior survival. Studies are ongoing toaddress the roles of ST3GAL6 over expression and altered sialylation inMM cell adhesion and trafficking.

The present inventors have shown that this glycogen is overexpressed inmultiple myeloma cell lines and in CD138 cells from myeloma patientswhen compared to their healthy counterparts. We have also demonstratedan association between increased expression of this gene and reducedmedian survival following analysis of a publically availabletranscriptomic dataset.

Altered glycosylation gene expression patters may identify patients athigh risk of disease progression and early death. Our data implicatessialytransferases and selectin ligands as potential therapeutic targetsin Multiple Myeloma. In particular, low expression of the FUCA1 gene isan adverse prognostic factor in MM and when combined with highsialyltransferase gene expression identifies patients at increased riskof early disease progression and death.

REFERENCES

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The words “comprises/comprising” and the words “having/including” whenused herein with reference to the present invention are used to specifythe presence of stated features, integers, steps or components but doesnot preclude the presence or addition of one or more other features,integers, steps, components or groups thereof.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination.

1. A method of determining the prognosis of a multiple myeloma patientor diagnosing multiple myeloma in a patient the method comprising thesteps of: determining the levels or activity of at least one biomarkerin a sample obtained from the patient, wherein the biomarker comprises(i) ST3GAL6, (ii) FUCA1, (iii) ST3GAL1, or (iv) a combination thereof,and comparing the level or activity of the biomarker in the sample withthe level or activity of the biomarker in a control sample.
 2. Themethod as claimed in claim 1, wherein the biomarker comprises FUCA1, andwherein a decreased level or activity of FUCA1 in the sample relative tothe control sample is predictive of a reduced survival rate or indicatesthe presence of multiple myeloma in the patient.
 3. The method asclaimed in claim 1, wherein the biomarker comprises ST3GAL1, and whereinan increased level or activity of ST3GAL1 in the sample relative to thecontrol sample is predictive of a reduced survival rate or indicates thepresence of multiple myeloma in the patient.
 4. The method of claim 1,wherein the biomarker comprises ST3GAL6, and wherein an increased levelor activity of ST3GAL6 in the sample relative to the control sample ispredictive of a reduced survival rate or indicates the presence ofmultiple myeloma in the patient. 5-6. (canceled)
 7. The method asclaimed in claim 1 wherein the diagnosis is of monoclonal gammopathy ofundetermined significance.
 8. A method of determining the treatmentregimen for a multiple myeloma patient comprising the steps of:determining the levels or activity of at least one biomarker in a sampleobtained from the patient, wherein the biomarker comprises (i) ST3GAL6,(ii) FUCA1, (iii) ST3GAL1, or (iv) a combination thereof, and comparingthe level or activity of the biomarker in the sample with the level oractivity of the biomarker in a control sample.
 9. The method as claimedin claim 8, wherein the biomarker comprises FUCA1, and wherein adecreased level or activity of FUCA1 in the sample relative to thecontrol sample indicates treatment with a FUCA1 potentiator would bebeneficial to the patient.
 10. The method as claimed in claim 8, whereinthe biomarker comprises ST3GAL1, and wherein an increased level oractivity of ST3GAL1 in the sample relative to the control sampleindicates treatment with a ST3GAL1 inhibitor would be beneficial to thepatient.
 11. The method as claimed in claim 1 wherein the sample is ablood, plasma, serum or tissue sample.
 12. The method of claim 1 whereinthe level of the biomarker is determined by immunohistochemistry,immunocytochemistry, RNA/DNA analysis, an ELISA assay, a competitive orinhibition ELISA, a sandwich ELISA assay, a micro-array based assay, afunctionalised nanoparticle assay, QDots, F1 tags, electro sensors, aflow cytometry assay or a real-time chip-based assay assays.
 13. Themethod of claim 1, wherein the control sample is from at least onehealthy subject. 14-22. (canceled)
 23. A method of reducing oralleviating multiple myeloma in a patient comprising the step ofadministering a modulator of ST3GAL6 in an amount sufficient to decreaseST3GAL6 expression or activity, a modulator of ST3GAL1 in an amountsufficient to decrease ST3GAL1 expression or activity, and/or amodulator of FUCA1 in an amount sufficient to increase FUCA1 expressionor activity. 24-25. (canceled)
 26. The method as claimed in claim 23wherein the modulator is an interfering RNA specific for ST3GAL6 orST3GAL1, a ST3GAL6 or ST3GAL1 enzyme inhibitor or a glycosyltransferaseinhibitor.
 27. The method as claimed in claim 23 wherein the modulatoris a FUCA1 enzyme potentiator. 28-30. (canceled)
 31. The method of claim8, wherein the biomarker comprises ST3GAL6, and wherein an increasedlevel or activity of ST3GAL6 in the sample relative to the controlsample indicates treatment with a ST3GAL6 inhibitor would be beneficialto the patient.
 32. The method of claim 8, wherein the sample is ablood, plasma, serum or tissue sample.
 33. The method of claim 8,wherein the level of the biomarker is determined byimmunohistochemistry, immunocytochemistry, RNA/DNA analysis, an ELISAassay, a competitive or inhibition ELISA, a sandwich ELISA assay, amicro-array based assay, a functionalised nanoparticle assay, QDots, F1tags, electro sensors, a flow cytometry assay or a real-time chip-basedassay.
 34. The method of claim 8, wherein the control sample is from atleast one healthy subject.